Fuctional analysis of Crr1 (Copper response regulator 1), a global regulator of nurtritional copper homeostasis in C. reinhardtii

如果铜在高浓度下积累，它会对细胞产生毒性，但它是一种必需的微量营养素，因为它是催化氧化还原反应的酶的辅因子。因此，细胞内铜的浓度和分布必须进行微调。在铜限制条件下，几种酶在C. reinhardtii，其i）用于增加铜的获得或ii）是含铜蛋白质的功能等效替代物。这种铜缺乏诱导的表达需要在诱导基因的启动子区域中的特异性铜响应元件（CuRE）以及特异性结合CuRE的转录因子Crr 1（铜响应调节因子1）。Crr 1被预测具有Zn/DNA结合结构域，与最近发现的植物特异性SBP家族转录因子同源，以及三个锚蛋白重复序列，通常参与蛋白质-蛋白质相互作用。该项目有两个目标：首先从丰度/定位、不同金属与Crr 1的结合特异性及其对DNA结合的影响方面表征Crr 1。应确定与Crr 1上锚蛋白结构域相互作用的其他转录因子。本实验将从分子水平阐明铜诱导基因表达的机制。在第二个蛋白质组学为基础的方法，进一步影响Crr 1/铜缺乏症和Crr 1的靶基因应被确定，这可以补充微阵列数据。

Copper can become toxic for cells if it accumulates in high concentrations, but it is an essential micronutrient since it functions as a cofactor for enzymes that catalyze e.g. redoxreactions. Intracellular concentration and distribution of copper has therefore to be fine tuned. Under copper limiting conditions several enzymes are expressed in C. reinhardtii which i) act to increase copper acquisition or ii) are functionally equivalent substitutes for copper containing proteins. This copper deficiency induced expression requires specific copper responsive elements (CuRE) in the promotor region of the induced genes as well as the transcription factor Crr1 (copper response regulator 1), which specifically binds to the CuRE. Crr1 is predicted to have a Zn/DNA-binding domain, homologoues to the recently discovered plant specific SBP-family transcription factors, as well as three ankyrin repeats, generally involved in protein-protein interaction. This project has two goals: First to characterize Crr1 in terms of abundance/localization, binding specificity of different metals to Crr1 and its implication on DNA-binding. Additional transcription factors interacting with the ankyrin domain on Crr1 should be identified. The proposed experiments will elucidate the mechanism of copper induced gene expression on the molecular level. In a second proteomics based approach further implications on Crr1/copper deficiency and target genes for Crr1 should be identified which can complement microarray data.